Biomedical subjects

U Mayer

Publications and source records attributed to U Mayer.

At least 73 records · Page 4Linked to original sources

PubMed1994

Changes in calcium and collagen IV binding caused by mutations in the EF hand and other domains of extracellular matrix protein BM-40 (SPARC, osteonectin).

Recombinant expression in a human kidney cell-line was used to prepare mutant human BM-40 with deletions including the N-terminal acidic domain, a central alpha-helical domain and the C-terminal EF hand domain. Two putative EF hand motifs were altered by point mutations. Only elimination of the whole EF hand domain or its single disulfide bond decreased production and secretion indicating that the C-terminal region of BM-40 is essential for correct folding. Deletions in the alpha-helical domain changed a single disulfide bond in this domain and caused oligomerization. Several mutations resulted in significant conformational changes as shown by CD spectroscopy and epitope analysis. Fluorescence titration demonstrated a single high-affinity (Kd = 0.08 microM) calcium-binding site with a low dissociation rate constant. A Glu to Lys mutation in the -Z position of the C-terminal EF hand motif and lack of a stabilizing disulfide bridge caused a 30 to 100-fold reduction in calcium affinity, while an Asp to Lys mutation in the X position had only a small effect. Deletions in the alpha-helical domain caused an even more dramatic reduction in calcium binding and abolished calcium-dependent binding of BM-40 to collagen IV. Both binding properties are critically dependent on a conformational interaction between the EF hand and the alpha-helical domain, which contains the collagen-binding site.

Amino Acid Sequence↗

PubMed1994

Influence of nidogen complexed or not with laminin on attachment, spreading, and albumin and laminin B2 mRNA levels of rat hepatocytes.

Nidogen/entactin is a Mr = 150,000 glycoprotein which is present within basement membranes in a noncovalent stable complex with laminin. We have studied the effects of nidogen/entactin complexed or not with laminin on attachment, spreading, and functions of adult rat hepatocytes in primary culture. Freshly isolated hepatocytes attached on either recombinant or EHS-derived nidogen, although to a lesser extent than on laminin/nidogen complex, laminin, and E8 and P1 fragments of laminin. Hepatocytes bound on a nidogen fragment bearing the N-terminal and rod-like domains but not on either the N-terminal globules or the rod-like domain which contains a RGD sequence. Attachment of hepatocytes on nidogen and laminin/nidogen complex was inhibited by anti-beta 1 integrin antibodies. Hepatocytes remained rounded on nidogen and laminin, whereas they rapidly spread on laminin/nidogen complex and collagen IV. Nidogen, laminin, and laminin/nidogen complex transiently maintained high steady-state albumin mRNA levels in cultured hepatocytes, but a decrease in albumin mRNA content was observed after 24 h, independently of the substrates. Actinomycin D and cycloheximide treatment indicated that the transient effect of these substrates on albumin expression was related to post-transcriptional mechanisms. Laminin B2 mRNAs were not detectable in freshly isolated hepatocytes but were expressed in 4 h hepatocyte cultures. After 24 h, a dramatic increase in the steady-state level of laminin B2 mRNA was found in hepatocytes cultured on nidogen and laminin/nidogen complex. This effect was slightly prevented in hepatocytes plated on laminin. These results show that interactions of hepatocytes with nidogen/entactin in vitro result only in a transient modulation of hepatocyte functions.

Animals↗

PubMed1994

Role of mesenchymal nidogen for epithelial morphogenesis in vitro.

Recent biochemical studies suggested that the extracellular matrix protein nidogen is a binding molecule linking together basement membrane components. We studied its expression and role during development. By immunofluorescence and northern blotting, nidogen was found early during epithelial cell development of kidney and lung. Yet, in situ hybridization revealed that nidogen was not produced by epithelium but by the adjacent mesenchyme in both organs. Binding of mesenchymal nidogen to epithelial laminin may thus be a key event during epithelial development. This is supported by antibody perturbation experiments. Antibodies against the nidogen binding site on laminin B2 chain perturbed epithelial development in vitro in embryonic kidney and lung. Mesenchymal nidogen could be important for early stages of epithelial morphogenesis.

Animals↗

PubMed1993

Sites of nidogen cleavage by proteases involved in tissue homeostasis and remodelling.

The cleavage of recombinant mouse nidogen in its native form was examined with granule-stored proteases (leucocyte elastase, mast-cell chymase), blood proteases (thrombin, plasmin, kallikrein), matrix metalloproteinases (stromelysin, matrilysin, collagenases) and, for comparison, with trypsin and the endoproteinase Glu-C. More than 50 major cleavage sites were identified by Edman degradation of several large fragments and smaller peptides. The data show an almost exclusive localization of protease-sensitive sites to the flexible segment, connecting the N-terminal globular domains G1 and G2, and within the C-terminal, laminin-binding domain G3. Domains G1, G2 and the rod-like segment were much more stable against proteolysis. Kinetic analysis indicated a fast cleavage of several different sites in the link region followed by destruction of G3 but this was to some extent variable depending on the particular protease. Leucocyte elastase was identified as the most active protease in the cleavage of nidogen whilst stromelysin, matrilysin, plasmin and kallikrein were of distinctly lower activity. No cleavage could be detected with interstitial collagenase and gelatinase A. The peptide analyses also allowed the location of two disulfide bridges within the G3 domain. Complex formation between nidogen and laminin fragments caused some protection against cleavage by thrombin, leucocyte elastase and stromelysin particularly in domain G3. The data indicate a relatively uniform cleavage pattern of nidogen which may be relevant in the context of protein/ligand-binding activities associated with domains G2 and G3. The proteolytic processes involved in remodelling and the cellular penetration of basement membranes could therefore be essential for the modulation of the mediator function of nidogen.

Animals↗

PubMed1993

Sequence of extracellular mouse protein BM-90/fibulin and its calcium-dependent binding to other basement-membrane ligands.

Partial sequence comparisons have recently indicated that two extracellular components, fibulin from human placenta and BM-90 from a basement-membrane-producing mouse tumor, are either identical or closely related proteins. In this study, a complete sequence analysis of mouse BM-90 cDNA showed a 539-residue N-terminal core structure (domains I and II), which was 85% identical with the same core structure of human fibulin. A 137-residue C-terminal sequence (domain III) was unique for BM-90 and could also be identified by Edman degradation. This suggested a novel splice product, variant D, which is characteristic for the mouse tumor. A second 117-residue C-terminal sequence (domain III) was identified in additional mouse cDNA clones and showed 91% identity with the region specific for variant C of fibulin. Northern blots using mouse cells demonstrated two mRNA species, 2.7 kb and 2.3 kb, which encoded the variants D and C, respectively. The sequence of BM-90/fibulin indicates the presence of nine epidermal-growth-factor-like repeats in the core domain-II structure, eight of which contain consensus motifs for calcium binding. This binding is apparently important for the interaction of BM-90 with laminin and nidogen and for some weaker interactions with collagen IV. Further binding of BM-90 was demonstrated to fibronectin and BM-90 itself, but did not depend on calcium. Major binding sites for BM-90 were identified at a C-terminal segment of laminin A chain and at the N-terminus of nidogen. The broad interaction repertoire of BM-90 is comparable to that of nidogen and both proteins may have similar roles as connecting elements in the extracellular matrix.

Alternative Splicing↗

PubMed1993

A single EGF-like motif of laminin is responsible for high affinity nidogen binding.

A major nidogen binding site of mouse laminin was previously localized to about three EGF-like repeats (Nos 3-5) of its B2 chain domain III [M. Gerl et al. (1991) Eur. J. Biochem., 202, 167]. The corresponding cDNA was amplified by polymerase chain reaction and inserted into a eukaryotic expression vector tagged with a signal peptide. Stably transfected human kidney cell clones were shown to process and secrete the resulting fragment B2III3-5 in substantial quantities. It possessed high binding activity for recombinant nidogen in ligand assays, with an affinity comparable with that of authentic laminin fragments. In addition, complexes of B2III3-5 and nidogen could be efficiently converted into a covalent complex by cross-linking reagents. Proteolytic degradation of the covalent complex demonstrated the association of B2III3-5 with a approximately 80 residue segment of nidogen domain G3 to which laminin binding has previously been attributed. The correct formation of most of the 12 disulfide bridges in B2III3-5 was indicated from its protease resistance and the complete loss of cross-reacting epitopes as well as of nidogen-binding activity after reduction and alkylation. Smaller fragments were prepared by the same recombinant procedure and showed that combinations of EGF-like repeats 3-4 and 4-5 and the single repeat 4 but not repeats 3 or 5 possess full nidogen-binding activity. This identifies repeat 4 as the only binding structure. The sequence of repeat 4 is well conserved in the human and in part in the Drosophila laminin B2 chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Nidogen mediates the formation of ternary complexes of basement membrane components.

Using a recombinant nidogen we have probed the calcium binding potential of various nidogen domains, examined the binding of nidogen to various basement membrane proteins and assessed the ability of nidogen to mediate the formation of ternary complexes between laminin and heparan sulfate proteoglycan and collagen IV and laminin. The results of these experiments indicate that the Ca+2 binding is on the rod-like domain with additional binding observed on the N-terminal G1 domain. With regard to the role of nidogen in mediating complex formation among basement membrane components it was demonstrated that nidogen effectively promotes the formation of a ternary complex between laminin and collagen IV, with both of these components interacting independently with nidogen. Similarly, nidogen mediates a ternary complex formation between laminin and proteoglycan. Interestingly, the interaction between proteoglycan and nidogen is through the protein core of the proteoglycan. We have localized the major interaction sites on nidogen with the proteoglycan core and collagen IV to a region on the globular G2 domain while the C-terminal globe G3 binds to laminin. Ca+2 binding does not appear to be important in either of the binary or ternary complex formations. The data reported allow us to hypothesize that, via the multiple interactions of nidogen with other basement membrane components, nidogen plays a crucial structural role in basement membrane organization and stabilization.

Animals↗

PubMed1993

Retinal phakomata associated with cerebral astrocytoma. An incomplete form of Bourneville-Pringle disease?

We report on a 21-year-old, male patient with unilateral retinal phakomata associated with histologically proved cerebral astrocytoma. The patient had presented with bilateral loss of vision and a left-sided hemiparesis. Ophthalmoscopy showed bilateral optic nerve atrophy, multiple punched-out areas of depigmentation and astrocytic hamartomata in the right eye. Despite the absence of classic signs of Bourneville-Pringle disease such as adenoma sebaceum, epilepsy and mental retardation, a strongly presumptive diagnosis of tuberous sclerosis could be made. This unusual case demonstrates that retinal phakomata can be the solely visible manifestation of Bourneville-Pringle disease.

Adult↗

PubMed1993

Structural basis of beta 1 integrin-mediated cell adhesion to a large heparan sulfate proteoglycan from basement membranes.

In a cell attachment assay, several cell lines were found to adhere and spread on heparan sulfate proteoglycan purified from a basement membrane-producing tumor. This adhesion was clearly distinct from that on laminin. Cell adhesion to the proteoglycan was completely inhibited by three different antibodies against integrin beta 1 subunit, while inhibitory antibodies against beta 3 and alpha 2 to alpha 6 subunits were without strong effects. Removal of heparan sulfate from the proteoglycan diminished cell attachment, but addition of heparin to the cells did not affect adhesion to the proteoglycan. This suggests that both the heparan sulfate side chains and core protein structures are required for efficient cell adhesion. Studies with proteolytic fragments and synthetic RGD peptides showed that the single RGD sequence of mouse proteoglycan is not involved in cellular recognition. Characterization of fragments also allowed to localize cell adhesion and heparan sulfate attachment sites to the same 160 kDa core protein structure but not to fragments derived from the N-terminal portion of the proteoglycan.

Amino Acid Sequence↗

PubMed1992

Basement-membrane heparan sulfate proteoglycan binds to laminin by its heparan sulfate chains and to nidogen by sites in the protein core.

A large, low-density form of heparan sulfate proteoglycan was isolated from the Engelbreth-Holm-Swarm (EHS) tumor and demonstrated to bind in immobilized-ligand assays to laminin fragment E3, collagen type IV, fibronectin and nidogen. The first three ligands mainly recognize the heparan sulfate chains, as shown by inhibition with heparin and heparan sulfate and by the failure to bind to the proteoglycan protein core. Nidogen, obtained from the EHS tumor or in recombinant form, binds exclusively to the protein core in a heparin-insensitive manner. Studies with other laminin fragments indicate that the fragment E3 possesses a unique binding site of laminin for the proteoglycan. A major binding site of nidogen was localized to its central globular domain G2 by using overlapping fragments. This allows for the formation of ternary complexes between laminin, nidogen and proteoglycan, suggesting a key role for nidogen in basement-membrane assembly. Evidence is provided for a second proteoglycan-binding site in the C-terminal globule G3 of nidogen, but this interaction prevents the formation of such ternary complexes. Therefore, the G3-mediated nidogen binding to laminin and proteoglycan are mutually exclusive.

Basement Membrane↗

PubMed1992

High-affinity and low-affinity calcium binding and stability of the multidomain extracellular 40-kDa basement membrane glycoprotein (BM-40/SPARC/osteonectin).

Reversible binding of calcium ions to a single high-affinity binding site in the 40-kDa basement membrane protein (BM-40) caused a 33% increase of alpha-helicity, an about 60% change in intrinsic fluorescence and a dramatic increase of the rate of cleavage by alpha-chymotrypsin. All these effects exhibited identical dependencies on calcium concentration from which a dissociation constant Kd = 0.6 microM was determined. Calcium release was accompanied by an increase of the frictional ratio in solution but not by denaturation which occurred at about equal guanidine.HCl concentration for both calcium-saturated and calcium-depleted protein (midpoint 1.5 M). The cleavage sites for alpha-chymotrypsin are located in or near to the EF-hand domain IV of calcium-depleted BM-40 (also known as SPARC, i.e. secreted protein acidic and rich in cysteine, and osteonectin). These and other data indicate that binding occurs in the EF-hand domain from which a large conformational change is transmitted. Low-affinity calcium-binding sites in the N-terminal glutamic-acid-rich domain I of BM-40 were identified by human leukocyte elastase which was found to cleave very specifically in the middle of this domain. From the increase of cleavage rate with increasing calcium concentration a Kd greater than or equal to 10 mM was estimated. It is suggested that variations of calcium levels in the extracellular space in this range may regulate functions of BM-40 such as collagen binding and that high-affinity binding is important for stabilization, folding and secretion during biosynthesis.

Amino Acid Sequence↗

PubMed1992

Structure of recombinant N-terminal globule of type VI collagen alpha 3 chain and its binding to heparin and hyaluronan.

A large portion of the N-terminal globule of human collagen VI was prepared from the culture medium of stably transfected human embryonic kidney cell clones. The recombinant product corresponds to sequence positions 1-1586 of the alpha 3 (VI) chain that consists of eight homologous approximately 200 residue motifs (N9 to N2) being similar to the A domain motif of von Willebrand factor. By ultracentrifugation fragment N9-N2 showed a molecular mass of 180 kDa and an asymmetric shape. Elongated structures that consist of eight small globes (diameter approximately 5 nm) were demonstrated by electron microscopy. The data indicate that each A domain motif represents a separate folding unit which are connected to each other by short protease-sensitive peptide segments. Circular dichroism studies demonstrated about 38% alpha helix, 14% beta sheets and 17% beta turns. Fragment N9-N2 showed binding to heparin which could be abolished by moderate salt concentrations. Heparin binding was assigned to domains N9, N6 and N3 which were obtained after partial proteolysis. Domains N7, N5 and N4 lacked affinity for heparin. In addition, N9-N2 showed strong binding to hyaluronan that required exposure to 6 M urea for full dissociation. Ligand binding studies indicated some affinity of N9-N2 for the triple helical region of collagen VI suggesting a role of the N-terminal globule in the self-assembly of microfibrils. No or only little binding was, however, observed to fibril-forming collagens I and III, several basement membrane proteins and other extracellular proteins. Fragment N9-N2 was also an inactive substrate for cell adhesion.

Amino Acid Sequence↗

PubMed1992

SUPERMAN, a regulator of floral homeotic genes in Arabidopsis.

We describe a locus, SUPERMAN, mutations in which result in extra stamens developing at the expense of the central carpels in the Arabidopsis thaliana flower. The development of superman flowers, from initial primordium to mature flower, is described by scanning electron microscopy. The development of doubly and triply mutant strains, constructed with superman alleles and previously identified homeotic mutations that cause alterations in floral organ identity, is also described. Essentially additive phenotypes are observed in superman agamous and superman apetala2 double mutants. The epistatic relationships observed between either apetala3 or pistillata and superman alleles suggest that the SUPERMAN gene product could be a regulator of these floral homeotic genes. To test this, the expression patterns of AGAMOUS and APETALA3 were examined in superman flowers. In wild-type flowers, APETALA3 expression is restricted to the second and third whorls where it is required for the specification of petals and stamens. In contrast, in superman flowers, APETALA3 expression expands to include most of the cells that would normally constitute the fourth whorl. This ectopic APETALA3 expression is proposed to be one of the causes of the development of the extra stamens in superman flowers. The spatial pattern of AGAMOUS expression remains unaltered in superman flowers as compared to wild-type flowers. Taken together these data indicate that one of the functions of the wild-type SUPERMAN gene product is to negatively regulate APETALA3 in the fourth whorl of the flower. In addition, superman mutants exhibit a loss of determinacy of the floral meristem, an effect that appears to be mediated by the APETALA3 and PISTILLATA gene products.

Gene Expression Regulation↗

PubMed1992

Pharmacodynamic and pharmacokinetic aspects of iodo-doxorubicin.

Iodo-doxorubicin (I-DOX) is a new doxorubicin (DOX) analog presently in phase II clinical trials in Europe. This drug is similar to DOX in its metabolism but differs in its pharmacokinetics. We describe correlations between pharmacokinetic parameters and toxicity. There is a linear relationship between the dose in mg/m2 and the nadirs of the reduction in white blood cells (WBC), neutrophils (PMN), platelets (PLT), and hemoglobin (HB). Furthermore, a linear correlation exists between the dose in mg/m2 and the area under the curve (AUC) of I-DOX. The relationship between the AUC of I-DOX and the reduction of PMNs plotted on a logarithmic scale shows a sigmoidal curve, whereas no such correlation was found between I-doxorubicinol, the major cytostatic metabolite, and the reduction in PNMs. The variation in the AUC for I-DOX correlated with the relative amount of metabolism of I-DOX. A correlation existed between the relative amount of metabolism of I-DOX and the reduction in PNMs. These results may have an impact on the evaluation of I-DOX in phase II trials, since a sound understanding of the pharmacokinetic/pharmacodynamic relationships can yield important information for minimizing unacceptable toxicity during phase II, as well as phase III, trials.

Biotransformation↗

PubMed1991

Recombinant expression and properties of the human calcium-binding extracellular matrix protein BM-40.

A cDNA construct (approximately 1 kb) of human BM-40 in a plasmid with the cytomegalovirus promoter and enhancer was used to produce several stable clones by transfecting two human cell lines (293, HT 1080). These clones showed a high expression of exogenous 1-kb BM-40 mRNA and no or only little endogenous 2.2-kb mRNA. These clones also secreted BM-40 at high rates (5-50 micrograms ml-1 day-1) into serum-free culture medium as shown by electrophoresis, radioimmunoassay and metabolic labelling. Transfection with the plasmid and overexpression of BM-40 had no effect on cell spreading, proliferation rate and adhesion patterns to extracellular matrix substrates. Recombinant human BM-40 was purified by anion-exchange chromatography and showed the expected N-terminal sequence and amino acid composition. The protein was also identical or similar to authentic BM-40 purified from the mouse Engelbreth-Holm-Swarm tumor in hexosamine content, electrophoretic mobility, circular dichroism and binding activity for calcium and collagen IV. Reduction of both authentic and recombinant BM-40 decreased binding activity which indicates correct formation of disulfide bonds in the recombinant protein. A specific and sensitive radioimmunoassay for human BM-40 was shown to be useful for detecting small quantities of the protein in human cell culture medium and blood. No significant cross-reaction was, however, detected between human and mouse BM-40.

Amino Acid Sequence↗

PubMed1991

Calcium-dependent binding of basement membrane protein BM-40 (osteonectin, SPARC) to basement membrane collagen type IV.

Basement membrane protein BM-40, prepared from the mouse Engelbreth-Holm-Swarm tumor, was used in native, denatured and proteolytically processed form for binding to various extracellular matrix proteins. BM-40 and its derivatives were also characterized by CD spectroscopy, calcium binding and epitope analysis. Of several basement membrane proteins tested only collagen IV showed a distinct and calcium-dependent binding of BM-40 in an immobilized ligand assay. This interaction was specific as shown by a low activity of other collagen types (I, III, V, VI) in direct binding and competition assays. The binding was reduced or abolished by metal-ion-chelating or chaotropic agents, high salt and reduction of disulfide bonds in BM-40. Fragment studies indicated that domains III (alpha-helix) and/or IV (EF hand) of BM-40 possess the binding site(s) for collagen IV, while the N-terminal domains I and II provide the major antigenic determinants. A major BM-40-binding site on collagen IV was dependent on a triple-helical conformation and could be localized to a pepsin fragment from the central portion of the triple-helical domain, in agreement with electron microscopic visualization of BM-40--collagen-IV complexes.

Amino Acid Sequence↗

PubMed1991

Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV.

Recombinant mouse nidogen and two fragments were produced in mammalian cells and purified from culture medium without resorting to denaturing conditions. The truncated products were fragments Nd-I (positions 1-905) comprising the N-terminal globule and rod-like domain and Nd-II corresponding mainly to the C-terminal globule (position 906-1217). Recombinant nidogen was indistinguishable from authentic nidogen obtained by guanidine dissociation from tumor tissue with respect to size, N-terminal sequence, CD spectra and immunochemical properties. They differed in protease stability and shape indicating that the N-terminal domain of the more native, recombinant protein consists of two globules connected by a flexible segment. This established a new model for the shape of nidogen consisting of three globes of variable mass (31-56 kDa) connected by either a rod-like or a thin segment. Recombinant nidogen formed stable complexes (Kd less than or equal to 1 nM) with laminin and collagen IV in binding assays with soluble and immobilized ligands and as shown by electron microscopy. Inhibition assays demonstrated different binding sites on nidogen for both ligands with different specificities. This was confirmed in studies with fragment Nd-I binding to collagen IV and fragment Nd-II binding to laminin fragment P1. In addition, recombinant nidogen but not Nd-I was able to bridge between laminin or P1 and collagen IV. Formation of such ternary complexes implicates a similar role for nidogen in the supramolecular organization of basement membranes.

Animals↗

PubMed1991

Human lens epithelial cells in tissue culture.

In 94 cultures of human cataractous lens epithelial cells derived from patients of different age, the cell diameter and morphology were observed and photographed after 21 days. The following results were obtained: every culture had its typical pattern depending on the age of the patient, which was from 3 months to 94 yr. The cell culture taken from a young man was characterized by smaller epitheloid cells showing a distinctly visible nucleus; the homogeneity of the monolayer diminished in parallel to the increasing age of the patients. The cell morphology became increasingly inhomogeneous, showed polymorphism and signs of vacuolic degeneration, and average cell diameter increased in parallel with the increasing age. The cells showed three characteristic phenotypes (Fig. 9), the morphology depending significantly on the age of the culture and of the donor: small epithelial-like cells without vacuoles showing a distinctly visible nucleus (most of these had a diameter of 50-100 microns), large cells with vacuoles, and cells without a visible nucleus and in rare cases including vacuoles (they appeared round and irregular, their diameter was 200-300 microns, and they included filamentous structures). Long term cultures were maintained for 8 months and subcultures maintained up to the third passage.

Adolescent↗